Annexin 5 pi double staining kit

For cell cycle analysis, cells were harvested and fixed in 70% ice-cold ethanol at 4°C overnight and then incubated with 100 μg/ml RNase at 37°C for 20 min. After staining with 50 μg/ml propidium iodide, cell cycle analysis was performed by fluorescence flow cytometry on a FACScan machine (Beckman Instruments). For apoptotic analysis, cells were washed and stained using an Annexin V/PI double staining kit (BD Biosciences) according to the manufacturer’s protocol.

To assess cell cycle progression, cells were added to 6-well plates (3× 10⁵/well) and cultivated for 24 h, at which time they were harvested using trypsin, rinsed with PBS, and fixed for 24 h with 70% ethanol at 4°C. Cells were subsequently stained with an AnnexinV/PI double staining kit (BD, USA) based on provided directions, followed by analysis via flow cytometry (BD, USA). Apoptotic cell death was assessed by plating cells under the same conditions, and then harvesting and rinsing these cells three times using PBS prior to resuspension in 100 uL of binding buffer. Cells were next stained with 5 uL of Annexin V-FITC and 10 uL of PI for 15 min on ice, after which 400 uL of binding buffer was added. Cells were analyzed within 1 h via flow cytometry (BD, USA).

Apoptotic induction of ADMSCs-TRAIL on solid tumour lines was performed using the annexin v/pi double staining kit purchased from (Becton Dickinson BD). For the analysis, direct co-cultured between both ADMSCs-TRAIL and tumour cells were performed using 24 wells plate, 0.4 μm transwell system for physical separation of both tumours and ADMSCs-TRAIL (SPL Life Sciences). Briefly, 1.0 × 10⁵ cells/well of ADMSCs-TRAIL and ADMSCs were seeded on the insert and the same number of tumours lines (1.0 × 10⁵ cells/well) were seeded on the bottom wells and co-cultured directly. 48 hours later, tumour cells was harvested by trysinization and collected by centrifugation. Cell palette was suspended in 100 μl of 1× annexin v binding buffer (Becton Dickinson BD) and 5 μl of annexin-v-FITC was added. Antibody incubation was performed at 4°C for 20 minutes and 1 μl of PI was later added before FACS aquisition. Stained cells were subjected to flow cytometric analysis using FACS Calibur instrument (Becton Dickinson BD) and a total of 10,000 events were acquired and analyzed using Cell Quest software (Becton Dickinson BD).

Cell proliferation was evaluated using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Cells (2 × 10³) were seeded in 96-well plates and cultured for 1, 2, 3, 4 and 5 days after transfection. CCK-8 solution (10 μl) was added to each well, followed by incubation for 2 h. Absorbance was measured at 450 nm with a Microplate Autoreader (Bio-Rad, Hercules, CA). Experiments were repeated at least three times. For the colony formation assay, cells were seeded in 6-well plates at a density of 500 cells per well and cultured for two weeks. Colonies were stained with 1% crystal violet for 30 s after fixation with 4% paraformaldehyde for 5 min, photographed and counted. Experiments were repeated at least three times. For cell cycle analysis, cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well. At 48 h after transfection, cells were detached via trypsinization, washed three times with ice-cold PBS and resuspended in 80% ethanol for at least 8 h at −20°C. Cells were fixed and stained with 50 μg/ml propidium iodide (Keygen, Nanjing, China). Cell cycle distribution was analyzed using FACSCaliber (BD Bioscience, MA, USA). For apoptosis detection, transfected cells were stained with AnnexinV/PI double staining kit (BD biosciences, Bedford, MA) according to the manufacturer's protocol. Apoptotic cells were examined by flow cytometry.